The safety and efficacy of several life-saving protein therapeutics are compromised by unwanted immune responses. Currently there are no viable clinical options to reduce or reverse these responses. Our goal is to develop protein delivery strategies that improve therapeutic efficacy of protein drugs by reducing or reversing unwanted immune responses. During the previous project period, we observed that exposure of mice to protein therapeutics in the presence of phosphatidylserine (PS) resulted in an antigen specific immunological tolerance/hypo-responsiveness towards the protein, leading to our central hypothesis that PS is immune regulatory and has the capacity to convert immunogenic proteins into tolerogens. To fully realize the clinical potential of this observation, it is necessary to determine the structural characteristics of PS that contribute to its ability to convert an immunogen into a tolerogen (SA1), and to use this information to reduce (SA2) and reverse (SA3) unwanted immune responses to therapeutic proteins and improve their therapeutic efficacy. In SA1 we hypothesize that PS converts dendritic cells from an immunogenic to a tolerogenic phenotype, generating regulatory T-cells that in turn suppress antigen-specific T-cells and B-cells. We investigate the structural and biophysical determinants of PS that program dendritic cells to send tolerogenic over immunogenic signals. Since pre-exposure of mice to FVIII in the presence of PS significantly reduced their immune response during re-challenge with FVIII, in SA2, we investigate a similar pre-treatment approach with two other immunogenic therapeutic proteins to prevent or reduce unwanted immune responses to these proteins during therapy. Specifically we will test this approach with acid alpha glucosidase (GAA in Pompe disease, a lysosomal storage disorder) and Factor IX (in Hemophilia B, a bleeding disorder). We will determine the immune cells that are involved in the induction of tolerance, and establish the impact of such tolerance induction on the efficacy of these two replacement therapies. In SA3, we develop clinical strategies to reverse established responses to FVIII, FIX and GAA, and thereby improve the efficacy of treatments for these bleeding and lysosomal disorders. Since PS is exposed in outer layer of secreted vesicles such as apoptotic bodies and on exosomes released from cells, we propose and will test the hypothesis that lipid particles mimicking these natural vesicles will reverse the established immune responses to therapeutic proteins. Our goal in SA3 is the optimization of immunization protocol and testing of an artificial lipid particle tha mimics the secreted vesicles, to reverse the unwanted established immune responses. These studies are expected to lead to novel clinical strategies to reduce and reverse immune responses to therapeutic proteins and thereby improve the efficacy of several lifesaving protein-based therapies.